Variant polynucleotide for increasing L-proline production

ABSTRACT

The invention relates to mutated variants of the proB gene from coryneform bacteria, which encode γ-glutamyl kinase, and to processes for fermentative production of L-proline using bacteria which contain this mutation.

The present invention relates to an enzyme having γ-glutamyl kinaseactivity. More particularly, the present invention characterizes thoseenzymes which have a proteinogenic amino acid other than glycine atposition 149 of the amino acid sequence or a comparable position.

The enzymes having γ-glutamyl kinase activity are preferably employed inthe fermentative production of L-proline. The L-proline biosyntheticpathway, starting from glutamate, is depicted in scheme 1.

It is known that amino-acids can be produced by fermenting strains of,for example, coryneform bacteria, in particular Corynebacteriumglutamicum. Due to the great importance, efforts are continually beingmade to improve the production processes. Procedural improvements mayconcern measures relating to fermentation technology, such as, forexample, stirring or oxygen supply, or the composition of the nutrientmedia, such as, for example, sugar concentration during thefermentation, or the working-up to the product form, for example bymeans of ion exchange chromatography, or the intrinsic performanceproperties of the microorganism itself.

The performance properties of these microorganisms are improved byapplying methods of mutagenesis, selection and mutant choice. Thisresults in strains which are resistant to anti-metabolites orauxotrophic for metabolites important for regulation and produce aminoacids. A known anti-metabolite is the proline analogue3,4-dehydro-DL-proline (DHP).

For some years now, recombinant DNA methods have likewise been used forimproving L-amino acid-producing Corynebacterium strains by amplifyingindividual amino acid biosynthesis genes and investigating the effect onamino acid production.

The nucleotide sequence of the Corynebacterium glutamicum genome isdescribed, inter alia, in EP-A-1108790 and has also been deposited inthe National Center for Biotechnology Information (NCBI) database of theNational Library of Medicine (Bethesda, Md., USA) under accessionnumbers NC_(—)003450.2 and BX927148.1 to BX927157.1.

Sleator et al. report the possibility of causing overproduction inproline biosynthesis by mutating the Listeria monocytogenes proB-Gen(Appl. Environ. Microbiol. 2001, 67, 4560-5). The mutations specifiedtherein and referred to as successful concern proB genes coding forγ-glutamyl kinases which have the following mutations: V121I, A144V andE146K. In addition, mention is made of the fact that the regions inwhich these mutations occur correspond very well to the region alsoidentified in other organisms as a target region for advantageousmutations.

It was therefore the object of the present invention to make availablefurther mutated and, where appropriate, improved protein variants of aγ-glutamyl kinase, which may be employed advantageously in a technicalprocess for fermentative production of L-proline.

This object and other objects which are not specified in detail butwhich arise in an obvious way from the prior art are achieved by statingthe γ-glutamyl kinases of claim 1. Claim 2 focuses on preferred enzymesof this kind. Claim 3 and 4 relates to the nucleotide sequences encodingthese enzymes, respectively, while claim 5 focuses on recombinantlyproduced vehicles having the nucleotide sequences just mentioned. Claim5, finally, relates to a production process according to the inventionfor L-proline with the aid of the enzymes mentioned.

By providing a γ-glutamyl kinase (product of the proB gene) which has aproteinogenic amino acid other than glycine at amino acid position 149or a comparable position, the object set out is achieved particularlysurprisingly, albeit no less advantageously. Compared to the wild-typeenzymes, γ-glutamyl kinases having an appropriate mutation assist inproducing L-proline in an improved manner in a fermentative productionprocess. Using the methods according to the invention, it is possible toimprove the performance of the host organisms or of the fermentationprocess with respect to one or more of the parameters selected from thegroup of product concentration (product per volume), product yield(product formed per carbon source consumed) and product formation(product formed per volume and time) or else other process parametersand combinations thereof by at least 0.5%, at least 1%, at least 1.5% orat least 2%, based on the starting strain or parent strain or thefermentation process with the use of said enzymes.

Preference is given to providing γ-glutamyl kinases in which an aminoacid, preferably L-amino acid, selected from the group consisting ofLys, Asn, Arg, Ser, Thr, Ile, Met, Glu, Asp, Ala, Val, Gln, His, Pro,Leu, Tyr, Trp, Cys or Phe is present at the amino acid position asdefined according to the invention or a comparable position. (The aminoacids mentioned, including glycine, are also referred to asproteinogenic amino acids in the art.) Very particular preference isgiven to the substitution of glycine with L-aspartic acid at position149 (G149D) in the said enzyme. Most preference is given to a γ-glutamylkinase according to the invention, as specified above, which is 369±40,preferably ±20, more preferably ±10 and very particularly preferably +5amino acids or ±3 amino acids in length. Enzymes intrinsic to the host,called aminopeptidases, are known to be able to cleave the N-terminalamino acid methionine off the protein formed. It is furthermore knownthat cleaving off one (1) or two (2) and no more than three (3) aminoacids from the C terminus of the protein impairs the enzyme activityonly negligibly at most, if at all. However, the enzyme may increase inlength due to appending certain fusion proteins (see below).

The present invention also encompasses the amino acid sequence of SEQ IDNO.: 2, preferably 4, or a sequence which is at least 90% identicalthereto, with the sequences comprising the amino acid substitution ofglycine by another amino acid, in particular any of the preferred aminoacids stated, at position 149 or a comparable position. The inventionthus also encompasses those enzymes which have the abovementioneddegrees of identity at the amino acid level in comparison with SEQ IDNO.: 2, preferably 4. These enzymes may likewise originate from naturalsources. Alternatively, they may have been modified by recombinant DNAtechnology in such a way that the skilled worker may predict the enzymicactivity to be retained or essentially retained (cf. for exampleSambrook et al, “Molecular Cloning, A Laboratory Handbook”, 2nd edition1989, CSH Press, Cold Spring Harbor, Ausubel et al. “Current Protocolsin Molecular Biology”, John Wiley & Sons, NY 2001). Thus, amino acidswhich are not present at the active site and whose substitution by anamino acid “of the same kind” is, prima facie, not expected to result ina substantially altered three-dimensional structure may be substitutedby an amino acid “of the same kind”. It may be expected, for example,that certain amino acids with non-polar side chains (amino acids of thesame kind) can be substituted, e.g. isoleucine by valine, without thishaving a (substantial) influence on the biological or enzymic functionof the enzyme according to the invention, or on the enzymic activity.The skilled worker may, on the basis of his knowledge, reachcorresponding conclusions also for the substitution of other types ofamino acids (for example the replacement of basic amino acids with otherbasic amino acids or of amino acids with uncharged polar side chainswith other amino acids from this group).

Preference is likewise given to a γ-glutamyl kinase having at least theamino acid sequence corresponding to positions, or comparable positions,145 to 154, more preferably 130 to 169 and very particularly preferably110 to 189 of SEQ. ID NO.: 2, preferably 4, it being possible for thelength of the γ-glutamyl kinase amino acid sequence to correspond to thenumbers indicated above.

In a furthermore preferred embodiment, the enzymes according to theinvention additionally comprise at least one heterologous amino acidsection by which these polypeptides are characterized as fusionproteins. Examples of heterologous components of the fusion proteinaccording to the invention may be tags (e.g. His tag or Flag tag) whichmay be employed in the purification of the fusion proteins according tothe invention. In other embodiments, the heterologous components mayhave a separate enzymic activity. In such a case, the two enzymiccomponents are preferably connected by a linker, such as a flexibleglycine or glycine-serine linker of 6-10 amino acids in length, in orderto ensure functionality of the components. The term “heterologous”, asused herein, may mean, on the one hand, that the components of thefusion protein do not naturally occur together in a covalently linkedform and, on the other hand, that the components are derived fromdifferent species. Fusion proteins are usually prepared by means ofrecombinant DNA technology (see Sambrook et al., loc. cit.).

In a further embodiment, the present invention relates to a nucleotidesequence (nucleic acid sequence) encoding a γ glutamyl kinase accordingto the invention, as specified above. Accordingly, the invention alsorelates to replicable nucleotide sequences coding for the enzymeγ-glutamyl kinase, it being possible for the corresponding amino acidsequences encoded by these nucleotide sequences to have anyproteinogenic amino acid, except glycine, at position 149 or acomparable position.

The nucleotide sequences of the invention preferably encode a γ-glutamylkinase, with the corresponding, encoded amino acid sequence comprisingan amino acid selected from the group consisting of Lys, Asn, Arg, Ser,Thr, Ile, Met, Glu, Asp, Ala, Val, Gln, His, Pro, Leu, Tyr, Trp, Cys orPhe at position 149 or a comparable position. A nucleotide sequenceencoding a γ-glutamyl kinase which has an L-aspartic acid at position149 or a comparable position is very particularly preferred.

The invention likewise relates to replicable nucleic acid sequencesencoding a γ-glutamyl kinase which has the amino acid substitutionaccording to the invention at position 149 or a comparable position,with these sequences

a) being at least 70% identical to Seq. ID NO.: 1, preferably SEQ. IDNO.: 3, or

b) encoding a γ-glutamyl kinase according to the invention, which is369±40 amino acids in length, or

c) encoding a γ-glutamyl kinase according to the invention, which hasthe amino acid sequence of SEQ. ID No.: 2, preferably SEQ. ID NO.: 4, atleast in positions or comparable positions 145 to 154, or

d) encoding a γ-glutamyl kinase according to the invention, which hasthe amino acid sequence of SEQ. ID No.: 2, preferably SEQ. ID NO.: 4, atleast in positions or comparable positions 145 to 154 and hybridizesunder stringent experimental conditions to the nucleotide sequencecomplementary to SEQ. ID NO.: 1 or SEQ. ID NO.: 3, orf) replicable nucleotide sequence which encodes the enzyme according tothe invention, γ-glutamyl kinase, and whose base sequence comprisesadenine at position 446, as depicted in SEQ ID NO.: 3.

The scope of the claims preferably likewise comprises replicable nucleicacid sequences encoding γ-glutamyl kinases according to the invention,which are 369±20, more preferably ±10 and most preferably ±5 or ±3,amino acid residues in length.

The invention also relates to a nucleotide sequence as depicted in SEQ.ID NO.: 1, preferably 3. As already mentioned, the invention alsoencompasses those sequences which are at least 70% identical to thatsequence at the nucleotide level. An example of a nucleotide sequencewhich is at least 70% identical to that of SEQ ID NO.: 3 is shown in SEQID NO.: 5. SEQ ID NO.:6 shows the amino acid sequence of γ-glutamylkinase, encoded by SEQ ID NO.: 5.

The invention likewise relates to replicable nucleic acid sequencesencoding γ-glutamyl kinases which preferably include at least the aminoacid sequence corresponding to positions 130-169 or comparablepositions, and very particularly preferably positions 110 to 189 of SEQID NO: 2, preferably 4, and which hybridize under stringent experimentalconditions with the nucleotide sequence complementary to SEQ ID NO: 1 orSEQ ID NO.: 3.

The term “complementary” means according to the invention that thecomplementarity extends without gaps across the entire region of thenucleic acid molecule according to the invention. In other words,according to the invention, preference is given to complementarityextending 100% across the entire region of the sequence according to theinvention, i.e. from the 5′ terminus depicted to the 3′ terminusdepicted, in particular the coding region (cds). In further preferredembodiments, complementarity extends across a region of at least 19,preferably at least 21, successive nucleotides which preferably do notencode the active site of enzymic activity.

Preference is given to those nucleic acid sequences derived fromcoryneform bacteria, preferably Corynebacterium glutamicum. Nucleic acidsequences of genes or alleles, which are present in the population of aspecies, are also referred to as endogenous genes or alleles in the art.

The present invention further relates to recombinant (rec) vehicleshaving the nucleotide sequences according to the invention. Suitablevehicles are any embodiments considered for this purpose by the skilledworker, in particular vectors and host organisms.

Examples of host organisms which may be mentioned in this regard areyeasts such as Hansenula polymorpha, Pichia sp., Saccharomycescerevisiae, prokaryotes such as E. coli, Bacillus subtilis, coryneformbacteria such as Corynebacterium glutamicum, or eukaryotes such asmammalian cells, insect cells or plant cells. These organismsaccumulate, where appropriate already prior to the measures of thepresent invention, L-proline in their cells or in the fermentationmedium surrounding them. Thus, in this preferred embodiment, the hostaccording to the invention is a recombinant cell which has beentransformed or transfected with a nucleic acid sequence according to theinvention or a vector according to the invention (see below) or providedwith them by way of conjugation (the terms “transformation”,“transfection” and “conjugation” are used synonymously according to thepresent invention). Transformation and transfection, respectively, maybe carried out according to known methods, for example by means ofcalcium phosphate co-precipitation, lipofection, electroporation,particle bombardment or viral infection. The cell according to theinvention may contain the recombinant nucleic acid extrachromosomally orin a chromosomally integrated form. In other words, thetransfection/transformation may be a stable or a transient one. Theprocesses for cloning are well known to the skilled worker (Sambrook,J.; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: alaboratory manual, 2^(nd) ed., Cold Spring Harbor Laboratory Press, NewYork). E. coli strains may be utilised for the recombinant preparationand mutagenesis methods, which include inter alia: E. coli XL1 Blue, NM522, JM101, JM109, JM105, RR1, DH5α, TOP10, HB101, BL21 codon plus, BL21(DE3) codon plus, BL21, BL21 (DE3), MM294. Plasmids used inter alia forcloning the gene construct having the nucleic acid according to theinvention into the host organism are likewise known to the skilledworker (see also PCT/EP03/07148; see below). A host of the genusCorynebacterium, of which particular mention may be made, is the speciesCorynebacterium glutamicum which is known in the art. Examples of knownwild-type strains of the genus Corynebacterium are:

-   -   Corynebacterium glutamicum ATCC13032    -   Corynebacterium acetoglutamicum ATCC15806    -   Corynebacterium acetoacidophilum. ATCC13870    -   Corynebacterium effiziens DSM 44549    -   Corynebacterium melassecola ATCC17965    -   Corynebacterium thermoaminogenes FERM BP-1539    -   Brevibacterium flavum ATCC14067    -   Brevibacterium lactofermentum ATCC13869 and    -   Brevibacterium divaricatum ATCC14020.

Information regarding the taxonomic classification of strains of thisgroup of bacteria can be found, inter alia, in Kämpfer and Kroppenstedt(Canadian Journal of Microbiology 42, 989-1005 (1996)) and in U.S. Pat.No. 5,250,434. For some years now (Liebl et al., International Journalof Systematic Bacteriology 41(2), 255-260 (1991)), coryneform bacteriawith the species names “Brevibacterium flavum”, “Brevibacteriumlactofermentum” und “Brevibacterium divaricatum” have been classifiedunder the species Corynebacterium glutamicum. Coryneform bacteria withthe species name “Corynebacterium melassecola” likewise belong to thespecies Corynebacterium glutamicum.

Examples of L-proline-producing strains of coryneform bacteria are thestrains:

-   -   Brevibacterium lactofermentum NRRL B-11421,    -   Brevibacterium flavum NRRL B-11422,    -   Corynebacterium glutamicum NRRL B-11423,    -   Microbacterium ammoniaphilum NRRL B-11424,    -   Corynebacterium glutamicum ATCC 21157,    -   Corynebacterium glutamicum ATCC 21158,    -   Corynebacterium glutamicum ATCC 21159,    -   Corynebacterium glutamicum ATCC 21355,    -   Corynebacterium acetophilum FERM-P 4045,    -   Corynebacterium acetoacidophilum FERM-P 4962,    -   Arthrobacter citreus FERM-P 4963, and    -   Microbacterium ammoniaphilum FERM-P 4964,        all of which are described in U.S. Pat. No. 4,224,409 and U.S.        Pat. No. 4,444,885.

In the vectors according to the invention, the nucleic acid sequencesaccording to the invention are preferably operatively linked to anexpression control sequence so as to be able to be transcribed and,where appropriate, translated in a suitable host cell. Expressioncontrol sequences usually comprise a promoter and, where appropriate,further regulatory sequences such as operators or enhancers. It isfurthermore also possible for translation initiation sequences to bepresent. Suitable expression control sequences for prokaryotic oreukaryotic host cells are known to the skilled worker (see, for example,Sambrook et al., loc. cit.). The recombinant vector according to theinvention may furthermore also include usual elements such as an originof replication and a selection marker gene. Examples of suitablerecombinant vectors are plasmids, cosmids, phages, or viruses (see, forexample, Sambrook et al., supra).

Suitable plasmids are in principle any embodiments available for thispurpose to the skilled worker. Such plasmids may be found, for example,in the paper by Studier and co-workers (Studier, W. F.; Rosenberg A. H.;Dunn J. J.; Dubendroff J. W.; (1990), Use of the T7 RNA polymerase todirect expression of cloned genes, Methods Enzymol. 185, 61-89) or inthe brochures from Novagen, Promega, New England Biolabs, Clontech orGibco BRL. Further preferred plasmids and vectors may be found in:Glover, D. M. (1985), DNA cloning: a practical approach, Vol. I-III, IRLPress Ltd., Oxford; Rodriguez, R. L. and Denhardt, D. T (eds) (1988),Vectors: a survey of molecular cloning vectors and their uses, 179-204,Butterworth, Stoneham; Goeddel, D. V. (1990), Systems for heterologousgene expression, Methods Enzymol. 185, 3-7; Sambrook, J.; Fritsch, E. F.and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nded., Cold Spring Harbor Laboratory Press, New York.

Plasmids which may be used for cloning the gene constructs having thecontemplated nucleic acid sequences into the host organism in a veryparticularly preferred manner are or are based on: pUC18/19 (RocheBiochemicals), pKK-177-3H (Roche Biochemicals), pBTac2 (RocheBiochemicals), pCR (Invitrogen), pKK223-3 (Amersham Pharmacia Biotech),pKK-233-3 (Stratagene) or pET (Novagen). Other preferred plasmids arepBR322 (DSM3879), pACYC184 (DSM4439) and pSC101 (DSM6202), all of whichmay be obtained from the DSMZ-Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, Brunswick, Germany. Examples of preferred promotersare the T7 promoter, lac promoter, tac promoter, trp promoter, rhapromoter and ara promoter.

Preferably, the γ-glutamyl kinases according to the invention or thenucleic acids encoding them are overexpressed in coryneform bacteria,preferably of the genus Corynebacterium, particularly preferably of thespecies Corynebacterium glutamicum.

Overexpression means an increase in the intracellular concentration oractivity of the γ-glutamyl kinases according to the invention.

The overexpression measures increase the activity or concentration ofthe corresponding protein usually by at least 10%, 25%, 50%, 75%, 100%,150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, basedon the activity or concentration of the protein in the startingmicroorganism.

Overexpression may be achieved by increasing the copy number of thegenes or alleles according to the invention by at least one (1) copy orby mutating the promoter region and regulatory region or theribosome-binding site which is located upstream of the structural gene.Expression cassettes incorporated upstream of the structural gene act inthe same manner. In addition, inducible promoters make it possible toincrease expression during fermentative L-proline production. Measuresof extending the mRNA life-time likewise improve expression. The enzymeactivity is furthermore amplified by preventing degradation of theenzyme protein. The genes or gene constructs may be present either inplasmids with different copy numbers or in the chromosome in anintegrated and amplified form. Alternatively, the genes in question mayfurthermore be overexpressed by altering the media composition and theculturing process.

Plasmids which are replicated in coryneform bacteria are useful forincreasing the copy number of the proB alleles according to theinvention. Numerous known plasmid vectors such as, for example, pZ1(Menkel et al., Applied and Environmental Microbiology (1989) 64:549-554), PEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1(Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmidspHM1519, pBL1 or pGA1. Other plasmid vectors such as, for example, thosebased on pCG4 (U.S. Pat. No. 4,489,160), or pNG2 (Serwold-Davis et al.,FEMS Microbiology Letters 66, 119-124 (1990)) or pAG1 (U.S. Pat. No.5,158,891) may be used in the same manner. An overview overCorynebacterium glutamicum plasmid vectors can be found in Tauch et al.(Journal of Biotechnology 104(1-3), 27-40 (2003).

It is furthermore possible to increase the copy number by applying themethod of chromosomal gene amplification which has been described, forexample, by Reinscheid et al. (Applied and Environmental Microbiology60, 126-132 (1994)) in the context of duplication and amplification ofthe hom-thrB-operon. This method involves cloning the complete gene orallele into a plasmid vector which can be replicated in a host(typically E. coli) but not in C. glutamicum. Examples of suitablevectors are pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)),pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pGEM-T(Promega Corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman, Journalof Biological Chemistry 269:32678-84 (1994); U.S. Pat. No. 5,487,993),pCR®Blunt (Firma Invitrogen, Groningen, the Netherlands; Bernard et al.,Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf etal., Journal of Bacteriology 173:4510-4516 (1991)) or pBGS8 (Spratt etal., Gene 41: 337-342 (1986)). The plasmid vector containing the gene orallele to be amplified is subsequently transferred by means ofconjugation or transformation to the desired C. glutamicum strain. Themethod of conjugation is described in Schäfer et al. (Applied andEnvironmental Microbiology 60, 756-759 (1994)), for example.Transformation methods are described in Thierbach et al. (AppliedMicrobiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan(Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)), for example. Afterhomologous recombination by way of a “cross over” event, the resultingstrain contains at least two copies of the gene or allele in question.In order to increase the copy number by at least 1, 2 or 3, it is inparticular also possible to use the tandem-amplification method asdescribed in WO 03/014330 or the method of amplification by way ofintegration at a desired location, as described in WO 03/040373.

Mutagenesis methods described in the prior art are used for generatingthe amino acid substitution according to the invention in γ-glutamylkinases (e.g. SEQ ID NO: 2) and other proB mutations according to theinvention, characterized by an amino acid substitution at position 149.Suitable mutagenesis methods are any methods available for this purposeto the skilled worker.

Classical in-vivo mutagenesis methods using mutagenic substances suchas, for example, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) orultraviolet light may be used for mutagenesis. The mutagenized cellsare, where appropriate, subsequently applied to a minimal agarcontaining 3,4-dehydro-DL-proline at concentrations of approx. 0.5-1g/l, approx. 1-2 g/l or approx. 2-3 g/l. Individual mutants are isolatedand the nucleotide sequence of the proB gene or allele is determined,where appropriate after a previous cloning process. It is furthermorepossible to use for the mutagenesis in-vitro methods such as, forexample, treatment with hydroxylamine (Miller, J. H.: A Short Course inBacterial Genetics. A Laboratory Manual and Handbook for Escherichiacoli and Related Bacteria, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 1992) or mutagenic oligonucleotides (T. A. Brown:Gentechnologie für Einsteiger [genetic engineering for beginners],Spektrum Akademischer Verlag, Heidelberg, 1993; textbook by Knippers(“Molekulare Genetik” [molecular genetics], 6^(th) edition, Georg ThiemeVerlag, Stuttgart, Germany, 1995); Winnacker (“Gene und Klone” [genesand clones], VCH Verlagsgesellschaft, Weinheim, Germany, 1990); Hagemann(“Allgemeine Genetik” [general genetics], Gustav Fischer Verlag,Stuttgart, Germany, 1986)) or the polymerase chain reaction (PCR) asdescribed in the manual by Newton and Graham (PCR, Spektrum AkademischerVerlag, Heidelberg, Germany, 1994). They include, in particular,saturation mutagenesis, random mutagenesis, in-vitro recombinationmethods and site-directed mutagenesis (Eigen, M. and Gardiner, W.(1984), Evolutionary molecular engineering based on RNA replication,Pure Appl. Chem. 56, 967-978; Chen, K. and Arnold, F. (1991), Enzymeengineering for nonaqueous solvents: random mutagenesis to enhanceactivity of subtilisin E in polar organic media. Bio/Technology 9,1073-1077; Horwitz, M. and Loeb, L. (1986), Promoters Selected FromRandom DNA-Sequences, Proc Natl Acad Sci USA 83, 7405-7409; Dube, D. andL. Loeb (1989), Mutants Generated By The Insertion Of RandomOligonucleotides Into The Active-Site Of The Beta-Lactamase Gene,Biochemistry 28, 5703-5707; Stemmer, P. C. (1994), Rapid evolution of aprotein in vitro by DNA shuffling, Nature 370, 389-391 and Stemmer, P.C. (1994), DNA shuffling by random fragmentation and reassembly: Invitro recombination for molecular evolution. Proc Natl Acad Sci USA 91,10747-10751). The use of in-vitro methods involves amplifying the proBgene described in the prior art with the aid of the polymerase chainreaction, starting from total DNA isolated from a wild-type strain,cloning the said gene, where appropriate, into suitable plasmid vectorsand then subjecting the DNA to the mutagenesis process. Instructions onthe amplification of DNA sequences with the aid of the polymerase chainreaction (PCR) can be found by the skilled worker, inter alia, in themanual by Gait: oligonucleotide Synthesis: A Practical Approach (IRLPress, Oxford, UK, 1984) and in Newton and Graham: PCR (SpektrumAkademischer Verlag, Heidelberg, Germany, 1994). In the same way it isalso possible to use methods of in-vitro mutagenesis, as described, forexample, in the well-known manual by Sambrook et al. (Molecular Cloning,A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA, 1989). Corresponding methods are alsocommercially available in the form of “kits” such as, for example, the“QuikChange Site-Directed Mutagenesis Kit” from Stratagene (La Jolla,USA), described by Papworth et al. (Strategies 9(3), 3-4 (1996)).Suitable proB mutants are subsequently selected and investigated by themethods described above.

For production of L-proline, it may be advantageous, in addition tousing the γ-glutamyl kinases according to the invention, which may ormay not have been overexpressed or amplified, to amplify, in particularto overexpress, besides the said kinases, at the same time one or moreenzymes of proline biosynthesis. Preference is usually given to usingendogenous genes.

“Endogenous genes” or “endogenous nucleotide sequences” means the genesor nucleotide sequences and alleles present in the population of aspecies.

In this context, the term amplification describes the increase in theintracellular activity or concentration of one or more enzymes orproteins in a microorganism, which are encoded by the corresponding DNA,by, for example, increasing the copy number of the gene or genes, usinga strong promoter or using a gene or allele which encodes acorresponding enzyme or protein with high activity and, whereappropriate, combining these measures.

The amplification, in particular overexpression, measures increase theactivity or concentration of the corresponding enzyme or protein usuallyby at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, upto a maximum of 1000% or 2000%, based on that of the wild-type proteinor on the activity or concentration of the protein in the startingmicroorganism.

For producing L-proline it is thus possible, in addition to using thevariant of the proB gene according to the invention, to amplify, inparticular overexpress, one or more of the genes selected from the groupconsisting of

-   -   the gdh gene coding for glutamate dehydrogenase (EC 1.4.1.4),    -   the proA gene coding for γ-glutamyl-phosphate reductase (EC        1.2.1.41),    -   the proC gene coding for pyrroline-5-carboxylate reductase (EC        1.5.1.2), and    -   the ocd gene coding for ornithine cyclodeaminase (EC 4.3.1.12).

For production of L-proline it may furthermore be advantageous, inaddition to using mutants according to the invention of the proB gene,at the same time to attenuate, in particular reduce expression of, oneor more of the endogenous genes selected from the group consisting of

-   -   the ilvA gene coding for threonine deaminase (EC 4.2.1.16),    -   the putA gene coding for proline        dehydrogenase/pyrroline-5-carboxylate dehydrogenase (EC        1.5.99.8),    -   the sucA gene coding for 2-ketoglutarate dehydrogenase (EC        1.2.4.2),    -   the sucB gene coding for dihydrolipoamide succinyltransferase        (EC 2.3.1.61), and    -   the argD gene coding for acetylornithine aminotransferase (EC        2.6.1.11).

In this connection, the term “attenuation” describes the reduction orelimination of the intracellular activity or concentration of one ormore enzymes or proteins which are encoded by the corresponding DNA in amicroorganism by, for example, using a weak promoter or using a gene orallele which encodes a corresponding enzyme with low activity orinactivating the corresponding gene or enzyme or protein and, whereappropriate, combining these measures.

Attenuation may be achieved by reducing or eliminating either expressionof the genes or the catalytic or regulatory properties of the enzymeproteins. Both measures may be combined, where appropriate.

Gene expression may be reduced by a suitable culturing process or bygenetic modification (mutation) of the signal structures of geneexpression. Examples of signal structures of gene expression arerepressor genes, activator genes, operators, promoters, attenuators,ribosome-binding sites, the start codon and terminators. Information onthis can be found by the skilled worker, for example, in the patentapplication WO 96/15246, in Boyd and Murphy (Journal of Bacteriology170: 5949-5952 (1988)), in Voskuil and Chambliss (Nucleic Acids Research26: 3584-3590 (1998), in Pátek et al. (Microbiology 142: 1297-309 (1996)and Journal of Biotechnology 104: 311-323 (2003)) and in known textbooksof genetics and molecular biology such as, for example, the textbook byKnippers (“Molekulare Genetik”, 6^(th) edition, Auflage, Georg ThiemeVerlag, Stuttgart, Germany, 1995) or that by Winnacker (“Gene undKlone”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

An example of specific regulation of gene expression is the cloning ofthe gene to be attenuated to come under the control of a promoterinducible by adding metered amounts of IPTG(isopropyl-β-D-thiogalactopyranoside), such as, for example, the trcpromoter or the tac promoter. Useful vectors here are, for example, theEscherichia coli expression vector pXK99E (WO0226787; deposited inaccordance with the Budapest Treaty on 31 Jul. 2001 in DH5alpha/pXK99Eas DSM14440 with the Deutsche Sammlung für Mikroorganismen undZellkulturen (DSMZ, Brunswick, Germany)) or pVWEx2 (Wendisch, Ph. D.thesis, Berichte des Forschungszentrums Jülich, Jül-3397, ISSN0994-2952, Jülich, Germany (1997)), which enable the cloned gene to beexpressed in an IPTG-dependent manner in Corynebacterium glutamicum.

This method was employed, for example, in the patent WO02/26787 forregulated expression of the deaD gene by integrating the vectorpXK99EdeaD into the Corynebacterium glutamicum genome, and by Simic etal. (Applied and Environmental Microbiology 68: 3321-3327 (2002)) forregulated expression of the glyA gene by integration of the vectorpK18mobglyA′ in Corynebacterium glutamicum.

Another method of specifically reducing gene expression is the antisensetechnique which involves introducing into the target cells shortoligoribonucleotides or oligodeoxyribonucleotides or vectors forsynthesizing longer antisense RNA. There, the antisense RNA may bind tocomplementary sections of specific mRNAs and reduce their stability orblock translatability. An example of this can be found by the skilledworker in Srivastava et al. (Applied Environmental Microbiology 2000October; 66 (10): 4366-4371).

Mutations which result in a change or reduction in the catalyticproperties of enzyme proteins are known from the prior art; exampleswhich may be mentioned are the studies by Qiu and Goodman (Journal ofBiological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (BioscienceBiotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (Ph. D.thesis, Berichte des Forschungszentrums Jülich, Jül-2906, ISSN09442952,Jülich, Germany (1994)). Overviews can be found in known textbooks ofgenetics and molecular biology, for example that by Hagemann(“Allgemeine Genetik”, Gustav Fischer Verlag, Stuttgart, Germany, 1986).

Mutations which come into consideration are transitions, transversions,insertions and deletions. Depending on the effect of the amino acidsubstitution on the enzyme activity, reference is made to missensemutations or nonsense mutations. A nonsense mutation leads to at leastone stop codon being located in the coding region of the gene andconsequently to translation being terminated prematurely. Insertions ordeletions of at least one base pair in a gene lead to frame shiftmutations as a result of which incorrect amino acids are incorporated ortranslation is terminated prematurely. Deletions of one or more codonstypically lead to a total loss of enzyme activity. Instructions forgenerating such mutations belong to the prior art and can be found inknown textbooks of genetics and molecular biology such as the textbookby Knippers (“Molekulare Genetik”, 6^(th) edition, Georg Thieme Verlag,Stuttgart, Germany, 1995), that by Winnacker (“Gene und Klone”, VCHVerlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann(“Allgemeine Genetik”, Gustav Fischer Verlag, Stuttgart, 1986).

As a result of using the measures for achieving attenuation, theactivity or concentration of the corresponding protein is usuallylowered to from 0 to 75%, from 0 to 50%, from 0 to 25%, from 0 to 10%,from 0 to 5% or from 0 to 1%, of the activity or concentration of thewild-type protein or of the activity or concentration of the protein inthe starting microorganism.

The microorganisms prepared according to the invention, which arelikewise a subject-matter of the present invention, may be culturedcontinuously or discontinuously, in a batch process or a fed-batchprocess or a repeated fed-batch process, for the purpose of producingL-proline. An overview of known culturing methods is described in thetextbook by Chmiel (Bioprozesstechnik 1. Einführung in dieBioverfahrenstechnik [Bioprocess Technology 1. Introduction toBioprocess Technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren und periphere Einrichtungen[Bioreactors and Peripheral Equipment] (Vieweg Verlag,Brunswick/Wiesbaden, 1994)).

The culture medium to be used must suitably satisfy the requirements ofthe particular strains. Descriptions of media for culturing variousmicroorganisms are given in the manual “Manual of Methods for GeneralBacteriology” published by the American Society for Bacteriology(Washington D.C., USA, 1981).

The carbon source employed may be sugars and carbohydrates, such as, forexample, glucose, sucrose, lactose, fructose, maltose, molasses, starchand cellulose, oils and fats, such as, for example, soybean oil,sunflower oil, peanut oil and coconut oil, fatty acids such as, forexample, palmitic acid, stearic acid and linoleic acid, alcohols suchas, for example, glycerol and ethanol, sugar alcohols such as, forexample, ribitol or mannitol and organic acids such as, for example,acetic acid. These substances may be used individually or as mixtures.

The nitrogen source employed may be organic nitrogen-containingcompounds such as peptones, yeast extract, meat extract, malt extract,cornsteep liquor, soybean flour and urea, or inorganic compounds such asammonium sulphate, ammonium chloride, ammonium phosphate, ammoniumcarbonate and ammonium nitrate. The nitrogen sources may be usedindividually or as mixtures.

The phosphorus source employed may be phosphoric acid, potassiumdihydrogen phosphate or dipotassium hydrogen phosphate or thecorresponding sodium-containing salts. The culture medium mustfurthermore contain salts of metals, for example magnesium sulphate oriron sulphate, which are necessary for growth. Finally, essential growthsubstances such as amino acids and vitamins may be used in addition tothe abovementioned substances. In addition to this, suitable precursorsmay be added to the culture medium. The added substances mentioned maybe added to the culture in the form of a once-only mixture or fed in asuitable manner during the culture.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammoniaor aqueous ammonia, or acidic compounds such as phosphoric acid orsulphuric acid are employed in a suitable manner for controlling the pHof the culture. It is possible to use antifoams such as, for example,fatty acid polyglycol esters, for controlling foam formation. Suitablesubstances which act selectively, such as antibiotics, for example, maybe added to the medium in order to maintain the stability of plasmids.In order to maintain aerobic conditions, oxygen or oxygen-containing gasmixtures such as air, for example, are passed into the culture. Thetemperature of the culture is normally from 20° C. to 45° C., andpreferably from 25° C. to 40° C. The culture is continued until amaximum of L-proline has been formed or until the yield or productivityhas reached a desired optimal level. This objective is normally achievedwithin from 10 hours to 160 hours.

Methods for determining L-proline are disclosed in the prior art. Theanalysis may, for example, take place by means of anion exchangechromatography, followed by ninhydrin derivatization and detection at anappropriate wavelength, as described in Spackman et al. (AnalyticalChemistry, 30 (1958), 1190).

Accordingly, another embodiment of the present invention constitutes aprocess for producing L-proline by

a) fermenting host organisms which express or overexpress at least oneof the nucleotide sequences according to the invention and

b) isolating or collecting L-proline, where appropriate with componentsfrom the fermentation broth and/or the biomass.

Preference is given to employing a process for producing L-proline,which comprises the following steps:

a) fermenting coryneform bacteria which express or overexpress at leastone of the nucleotide sequences according to the invention,

b) concentrating L-proline in the fermentation broth or in the cells ofthe coryneform bacteria,

c) isolating or collecting L-proline from the fermentation broth, whereappropriate

d) with components from the fermentation broth and/or the biomass(from >0 to <100% by weight of biomass, preferably from 10 to 80% byweight, more preferably 20-60% by weight).

The L-proline produced in this way may be collected and isolated and,where appropriate, purified, as determined by the skilled worker.

The process according to the invention is used for fermentativeproduction of L-proline.

The term “comparable position” means according to the invention aposition which, by comparing the starting sequence with the comparativesequence with application of a sequence comparison program (BLAST,Altschul et al. J. Mol. Biol. 1990, 215, 403-10) at the comtemplatedposition of the starting sequence, provides an amino acid position inthe comparative sequence which differs from the position to be comparedby no more than ±5, more preferably ±4, further preferably ±3, stillfurther preferably ±2, most preferably ±1, and especially zero,positions.

Instructions regarding hybridization can be found by the skilled workerinter alia in the manual “The DIG System Users Guide for FilterHybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993)and in Liebl et al. (International Journal of Systematic Bacteriology41: 255-260 (1991)). The hybridization is carried out under stringentconditions, i.e. only hybrids in which the probe, for example thenucleotide sequence complementary to SEQ ID NO: 3, and the targetsequence, i.e. the polynucleotides treated with the probe, are at least70% identical, are formed. The stringency of the hybridization,including that of the washing steps, is known to be influenced ordetermined by varying the buffer composition, the temperature and thesalt concentration. The hybridization reaction is generally carried outat relatively low stringency compared to the washing steps (HybaidHybridisation Guide, Hybaid Limited, Teddington, UK, 1996).

For example, a buffer corresponding to 5×SSC buffer at a temperature ofapprox. 50° C.-68° C. may be used for the hybridization reaction. Inthis case, probes may also hybridize with polynucleotides which are lessthan 70% identical to the sequence of the probe. Such hybrids are lessstable and are removed by washing under stringent conditions. This maybe achieved, for example, by lowering the salt concentration to 2×SSCand, where appropriate, subsequently to 0.5×SSC (The DIG System User'sGuide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany,1995), with the temperature being set to approx. 50° C.-68° C., approx.52° C.-68° C., approx. 54° C.-68° C., approx. 56° C.-68° C., approx. 58°C.-68° C., approx. 60° C.-68° C., approx. 62° C.-68° C., approx. 64°C.-68° C., approx. 66° C.-68° C. Preference is given to carrying out thewashing steps at temperatures of approx. 62° C.-68° C., particularlypreferably approx. 64° C.-68° C. or approx. 66° C.-68° C. It ispossible, where appropriate, to lower the salt concentration down to aconcentration corresponding to 0.2×SSC or 0.1×SSC. By graduallyincreasing the hybridization temperature in steps of approx. 1-2° C.from 50° C. to 68° C., it is possible to isolate polynucleotidefragments which have at least 70% or at least 80% or at least 90% to 95%or at least 96% to 98% or at least 99% identity to the sequence of theprobe employed. Further instructions regarding hybridization arecommercially available in the form of “kits” (e.g. DIG Easy Hyb fromRoche Diagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558)).

According to the invention, the claimed polypeptides (amino acidsequences) and the nucleic acid sequences also comprise those sequenceswhich have a homology (at the amino acid level) and, respectively,identity (at the nucleic acid level, excluding natural degeneracy) ofmore than 70%, preferably 80%, more preferably 85% (with respect to thenucleic acid sequence) or 90% (also with respect to the polypeptides),preferably more than 91%, 92%, 93% or 94%, more preferably more than 95%or 96% and particularly preferably more than 97%, 98% or 99% (withrespect to both types of sequences) to any of these sequences, as longas the action or purpose of such a sequence is retained. The term“homology” (or identity), as used herein, can be defined by the equationH (%)=[1−V/X]×100, where H is homology, X is the total number ofnucleobases/amino acids of the comparative sequence and V is the numberof different nucleobases/amino acids of the sequence to be considered,based on the comparative sequence. In any case, the term nucleic acidsequences coding for polypeptides includes any sequences that seempossible in accordance with the degeneracy of the genetic code.

The percentage identity to the amino acid sequences indicated in thepresent specification by SEQ ID numbers may readily be determined by theskilled worker using methods known in the prior art. A suitable programwhich may be employed according to the invention is BLASTP (Altschul etal., 1997. Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs. Nucleic Acids Res: 25(17):3389-3402.). Thenucleic acid sequence according to the invention may be a DNA moleculeor an RNA molecule. Preference is given to the nucleic acid sequencebeing a DNA molecule or an mRNA molecule. According to the invention,the DNA molecule may furthermore be a genomic or an isolated DNAmolecule. The invention further encompasses embodiments in which the DNAmolecule is a PNA molecule or another derivative of a DNA molecule.

The microorganisms mentioned in this application, which are indicated bya DSMZ number, may be obtained from the Deutsche Sammlung fürMikroorganismen und Zellkulturen, Mascheroder Weg 4, Brunswick(Germany).

1. An isolated polynucleotide, which encodes a polypeptide comprising ofSEQ ID NO:
 4. 2. The isolated polynucleotide of claim 1, which comprisesSEQ ID NO:3.
 3. A recombinant vector comprising the isolatedpolynucleotide of claim
 1. 4. A recombinant vector comprising theisolated polynucleotide of claim
 2. 5. A host cell comprising theisolated polynucleotide of claim
 1. 6. A host cell comprising theisolated polynucleotide of claim
 2. 7. The host cell of claim 5, whichis a Corynebacterium host cell.
 8. The host cell of claim 6, which is aCorynebacterium host cell.